PROJECT SUMMARY/ABSTRACT The broad objectives of this proposal are to 1) foster the development of Dr. Panagiotis Flevaris as an independent academic investigator in in the field of cardiovascular medicine, and 2) to investigate the fundamental signaling mechanisms that initiate and accelerate cardiac fibrosis, a final common manifestation of nearly every form of heart disease. This program has been designed to help the PI gain additional skills through 1) graduate level coursework in the Interdisciplinary Biological Sciences program at Northwestern University, 2) development of expertise in new scientific methods (human induced pluripotent stem cell (hiPSC) biology), and 3) development of skills in experimental design and scientific communication through structured interdisciplinary mentorship and the peer review process. The primary mentor, Dr. Douglas Vaughan is a renowned cardiovascular investigator with a long track record of training physician scientists and with expertise in cardiac fibrosis, a cause of tremendous morbidity and mortality in the United States. Despite being an area of intense investigation, the only effective treatment for fibrotic cardiomyopathy is heart transplantation. In this proposal we will utilize a cardiomyocyte(CM)-specific PAI-1 knockout mouse model to replicate a novel fibrotic cardiomyopathy we recently uncovered in a large human kindred harboring a mutation in SERPINE-1 (the gene that encodes for PAI-1). Our preliminary data reveal that ventricular myocytes from global PAI-1 knockout mice generate significantly elevated levels of the profibrotic cytokine TGF-? in vitro and in vivo. We have also identified that PAI deficiency is results in reduced cardiac SMAD6, a negative regulator of TGF-? signaling. Furthermore, recombinant BMP7, a known inducer of SMAD6 expression, abrogates TGF-? synthesis in both hiPSC-derived and primary mouse CMs, and inhibits cardiac fibrosis during injury. Thus, we hypothesize that cardiomyocyte TGF-? drives cardiac fibrosis, and can be pharmacologically inhibited by BMP7-dependent SMAD6 induction. We examine this hypothesis with three specific Aims: 1) To define how CM-specific deletion of PAI-1 enhances cardiac TGF-? and its associated epigenetic and transcriptional signature during injury-mediated cardiac fibrosis, 2) To define how SMAD6 modulates hiPSC-CM TGF-? signaling and genetic programs in response to PAI-1 deficiency or injury, and 3) To utilize BMP7-dependent stimulation of SMAD6 in CMs to abrogate TGF- ? autocrine signaling, and the epigenetic and transcriptional changes that drive cardiac fibrosis. Parallel investigation of human CM and murine models that replicate the human disorder will fuel discoveries broadly relevant to fibrotic cardiomyopathies involving the TGF-? axis, and uncover novel approaches that could be translated for therapeutic benefit in a broad spectrum of patients with cardiac fibrosis.